JP2014508159A - GLA domain deletion factor XA for treating hemophilia A or B with or without inhibitors - Google Patents

Abstract

The present invention relates to a pharmaceutical composition comprising modified factor Xa (GDXa) for preventing or treating hemorrhagic accidents in patients with hemophilia A or B who do or do not have an inhibitor, comprising the modified GDXa Relates to a pharmaceutical composition that is non-thrombogenic and can bind to TFPI but cannot bind to phospholipids.
[Selection] Figure 5

Description

  The present invention relates to a pharmaceutical composition for preventing or treating hemorrhagic accidents in patients with hemophilia A or B with modified factor X.

  As with hemophilia B, hemophilia A includes two types of hemophilia, constitutional hemophilia and acquired hemophilia.

  Type A constitutional hemophilia is a hemorrhagic disease characterized by quantitative or qualitative dysfunction of FVIII caused by abnormalities in the FVIII gene. Type B constitutional hemophilia is also a hemorrhagic disease, but is characterized by quantitative or qualitative failure of FIX caused by abnormalities in the FIX gene.

  Acquired hemophilia of type A or B is defined by the appearance of autoantibodies specific for said FVIII or FIX.

  Hemophilia appears in blood coagulation failure in response to bleeding. Untreated type A or B hemophilia also has symptoms such as excessive bleeding at the time of injury and sometimes sudden bleeding.

  The biological activity of factor VIII or factor IX is assessed as a percentage of the normal level. Normal individuals are considered to have 100% activity. If the activity is not detectable (less than 1%), it is severe hemophilia; if the activity is 1-5%, this hemophilia is said to be moderate and above 5% to 30% mild blood. It is friendship.

  Hemophilia A and B patients can be treated with concentrates containing FVIII or FIX, respectively, which can be plasma derivatives or products produced by genetic engineering. These concentrates can be administered at the time of each bleeding, in which case it is desirable to begin treatment as soon as possible once the first signs appear. This treatment can also be administered prophylactically and regularly 2-3 times a week to prevent bleeding. However, this treatment can cause the appearance of antibodies specific to FVIII or FIX called inhibitors. The presence of the antibody then negates the administration of Factor VIII or Factor IX. These antibodies are G-type immunoglobulins, mainly IgG4. Antibodies appear shortly after the first dose, often before 10 days. Some patients maintain a weak responder (antibody titer <10 BU), while others called strong responders have a titer that means they can no longer be treated with the corresponding coagulation factor. Has reached.

  At the time of the present invention, there are no treatments that can sufficiently prevent and / or treat the presence of the risk of bleeding in hemophilia A or B patients carrying inhibitors. In fact, the available products may be invalid (for FSG, Astermark J, Donfield SM, DiMichel DM, Gringeri A, Gilbert SA, Waters J, Berntorp E in hemophilia with inhibitors A randomized comparison of bypassing agents in hemophilia complicated by an inhibitor ": the FEIBA NovoSeven Comparative (FENOC) Study. Blood. 2007; 109: 546-51), or administration of their products Can be complicated by thrombus formation events (Aledort LM. “The incidence of thrombogenic events after injection of recombinant factor Vila versus factor VIII inhibitor bypass actives” (. Comparative thrombotic event incidence after infusion of recombinant factor VIIa versus factor VIII inhibitor bypass activity) compared "J Thromb Haemost.2004; 2: 1709).

Therefore, it is known that there is a need for treatments that are alternatives to existing treatments. However, the development of such treatments has proven to be very difficult because
Can stop bleeding,
Does not cause thrombosis,
This is because it must be possible to treat or prevent bleeding accidents even in the presence of anti-FVIII or FIX antibodies.

  The present invention addresses this need. The present invention relates to a pharmaceutical composition comprising modified activated factor X (FXa) for preventing or treating hemorrhagic accidents in patients with hemophilia A or B, wherein said modified FXa (GDXa) is thrombogenic. Can bind to tissue factor pathway inhibitor (TFPI) but does not have a phospholipid binding site.

  The composition according to the invention can also be used to prevent or treat hemorrhagic accidents in haemophilia exhibiting anti-factor VIII (FVIII) or factor IX (FIX) antibodies. The antibody appeared after treatment with factor FVIII or factor FIX or spontaneously, eg in acquired hemophilia.

  The present invention also relates to the use of a pharmaceutical composition comprising modified factor Xa (GDXa) for preparing a pharmaceutical product intended for preventing or treating hemorrhagic accidents in patients with hemophilia A or B The patient has or does not have anti-FVIII or FIX antibodies.

  The invention also relates to a method of prophylactically treating hemorrhagic syndrome in patients with hemophilia A or B by administration of modified factor Xa (GDXa).

  The present invention also relates to a method of treating hemorrhagic accidents in hemophilia A or B patients by administration of modified factor Xa (GDXa).

  In the context of the present invention, factor FXa refers to an activated factor obtained by activation of native factor X, either naturally occurring in plasma or isolated in its original unmodified form. Means factor X. The term includes FX isolated from plasma, but also includes activated FX produced recombinantly or obtained by chemical synthesis methods. Factor Xa or native factor Xa (FXa), in the context of the present invention, is a serine protease type protein involved in blood coagulation and is produced in an inactivated form, factor X (FX) .

  Coagulation factor X activation is an important step in preventing blood clotting and bleeding. Its activation is necessary for the steps of blood coagulation propagation and amplification. Its activation is also necessary to prevent activation of blood coagulation by interaction with TFPI.

  FX is activated by either activated factor IX and its cofactor, activated factor VIII or activated factor VII and its cofactor, tissue factor (TF). FXa forms a prothrombinase complex that binds to the membrane with activated factor V and becomes the active component in the prothrombinase complex, which catalyzes the conversion of prothrombin to thrombin. Thrombin then catalyzes the conversion of fibrinogen to fibrin, which leads to the formation of blood clots and the prevention of bleeding. The activity of FXa can be referred to as “blood coagulation promoting activity”.

  Leytus et al. (Biochemistry, 1986, 25: 5098-5102) and Venkateswarlu et al. (Biophysical Journal, 2002, 82: 1190-1206) describe Factor X and the various domains present in this polypeptide. Catalytic cleavage of the heavy chain allows FX to FXa activation. FXa includes a light chain (one example represented by SEQ ID NO: 1) and a heavy chain (one example represented by SEQ ID SEQ ID NO: 2).

  In the context of the present invention, the term modified FXa refers to FXa that can no longer bind to phospholipids, no longer has procoagulant activity, or has reduced procoagulant activity. In the context of the present invention, such a factor is called GDXa. “Procoagulant activity” is defined as the ability of a factor to cause blood clotting or clot formation. Reduced blood coagulation promoting activity indicates that the activity is reduced by at least 50%, preferably at least 90%, even more preferably more than 95% compared to the activity of native FXa.

  The modified factor Xa, GDXa, according to the present invention lacks a γ-carboxyglutamate (Gla) domain for binding to phospholipids. The first 43 amino acids of the light chain (residues 1 to 43 of SEQ ID NO: 1) contain 11 post-translationally modified residues (γ-carboxyglutamic acid) and thus represent the Gla domain. Digestion with chymotrypsin can suppress residues 1-43 and produce FXa or GDXa (FXa without Gla domain) lacking a domain that binds to phospholipids. The GDXa can also include other modifications in addition to the absence of the Gla domain. The modified FXa preserves the binding property to factor Va, but does not have blood coagulation promoting activity. One example of GDXa is represented by SEQ ID NO: 7 or SEQ ID NO: 3 for the light chain and SEQ ID NO: 2 for the heavy chain. This lack of blood clotting activity can be confirmed by the inability to activate blood clotting when added to plasma without tissue factor, thereby distinguishing it from natural factor Xa. The

  GDXa can be obtained by cleavage of factor X by proteolysis controlled by chymotrypsin and activation by a specific protease according to one of the usual methods, for example, the method described by Skogen et al. (1984). An example of GDX prior to activation can be represented by the sequence identifier SEQ ID NO: 20 for the nucleic acid sequence and SEQ ID NO: 28 for the amino acid sequence.

  GDXa can be produced by chemical synthesis, either in the form of a single sequence or in the form of several sequences that are later joined together. This synthesis can be carried out in solid phase or in solution. These techniques are described in “Solid phase peptide synthesis” (IRL Press Oxford, 1989) by Atherton and Shepard and “Methoden der organischen Chemie” published by Houbenweil (E. Wunsch) [ Methods in Organic Chemistry] "Vol. 15-1 and 11, Stuttgart, 1974) and the following article: E. Dawson et al. (Science 1994; 266 (5186), pp 776-779); GG Kochendorfer et al. (1999; 3 (6), pp 665-671); P E Dawson et al. (2000, 69, Annu. Rev. Biochem., Pp 923). ~ 960).

  That GDXa has no thrombogenicity and no procoagulant activity can be confirmed by an assay that measures thrombin generation (Hemker et al., Thrombosis and Haemostasis. 1993; 70: 617-624, eg 4).

  The ability of GDXa to bind to TFPI is confirmed using any assay known to those skilled in the art. Such an assay is described in Example 1 and uses a chromogenic substrate that allows measurement of percent inhibition of FXa and GDXa by TFPI.

  In another aspect of the invention, GDXa lacks the Gla domain, but also lacks domain EGF1 (epidermal growth factor 1). Such an application of the invention can be represented by a composition comprising GDXa represented by SEQ ID NO: 4 for the light chain and SEQ ID NO: 2 for the heavy chain.

  In another aspect of the invention, GDXa lacks the Gla domain, but also lacks domain EGF2 (epidermal growth factor 2). One example of such GDXa is represented by SEQ ID NO: 5 for the light chain and SEQ ID NO: 2 for the heavy chain.

  In another aspect of the invention, GDXa lacks domain Gla, but also lacks domains EGF1 and EGF2. One example of such GDXa is represented by SEQ ID NO: 6 for the light chain and SEQ ID NO: 2 for the heavy chain.

  According to another aspect of the present invention, GDXa is composed only of the heavy chain of FXa. According to a particular aspect of the invention, such FXa is represented by the sequence identifier SEQ ID NO: 2.

  In yet another different aspect, GDXa is composed of molecular variants with mutations. Therefore, various mutations were introduced into the gene encoding GDXa that can maintain thrombin generation activity and reduce the enzyme activity of the mutant against small peptide substrates. These mutations can be introduced using the QuickChange kit (Stratagene) according to the manufacturer's recommendations according to the publication Wang and Malcolm (1999) -BioTechniques, 26: 680-682. These mutations can be related to GDXa heavy chain arginine 142 (numbered according to SEQ ID NO: 2), which arginine can be any other amino acid, preferably phenylalanine (eg, SEQ ID NO: 10), glycine ( For example, it can be mutated to make it SEQ ID NO: 11), isoleucine (eg SEQ ID NO: 12) or tyrosine (eg SEQ ID NO: 13). This mutation is derived from the bovine FXa: Arg-Leu-Ser-Ser-Thr-Leu (eg SEQ ID NO: 26) of the peptide sequence of human FXa Arg-Gln-Ser-Thr-Arg-Leu (heavy chain 139-143) Can be replaced with the equivalent sequence obtained from. Similarly, heavy chain lysine 82 (numbered according to SEQ ID NO: 2) can also be replaced with an amino acid, eg, tyrosine (eg, SEQ ID NO: 9).

  The nucleotide sequence encoding GDXa can be chemically synthesized (Young L and Dong Q., 2004, Nucleic Acids Res., Apr 15; 32 (7), Hoover, DM and Lubowski, J. et al. 2002. Nucleic Acids Res., 30, Villalobos A et al., 2006. BMC Bioinformatics, Jun 6; 7: 285). The nucleotide sequence encoding GDXa can also be amplified by PCR using appropriate primers.

  GDXA can also be produced by genetic engineering techniques well known to those skilled in the art. The nucleotide sequence encoding human factor X can thus be cloned into an expression vector, ie the nucleotide sequence encoding the signal peptide, the propeptide and part of the domain Gla are deleted and the signal peptide, eg TIMP- One signal peptide (Crombez et al., 2005) is fused. The modified factor X thus generated is either by the TF-FVIIa complex or any other enzyme that cleaves the bond between arginine 234 and isoleucine 235 (numbered according to Swiss plot: P00742.2). Can be activated. Alternatively, GDXa can be generated directly by inserting a cleavage sequence recognized by furin or any other intracellular enzyme immediately upstream of isoleucine 235. That is, sequences encoding these amino acids, eg, arginine-lysine-arginine, allow cleavage by furin (Nakayama et al., 1997). To improve cleavage, the sequence arginine-lysine-arginine-arginine-lysine-arginine can be introduced. The DNA encoding the modified FX is inserted into an expression plasmid and inserted into a special cell line (eg, HEK-393E system) to produce them, and the protein thus produced is then purified by chromatography. The

  These techniques are described in detail in the reference manuals: Molecular cloning: a laboratory manual, 3rd edition, edited by Sambrook and Russell (2001) and Current Protocols in Molecular Biology-Ausubel et al. (2007).

  Thus, GDXa can also be represented by the nucleotide sequence encoding GDXa described above, and such a sequence is represented by the following sequence identifiers: SEQ ID NO: 8, SEQ ID NO: 16 to SEQ ID NO: 19, heavy chain The chain is represented by SEQ ID NO: 15 or SEQ ID NOs: 21-25 and SEQ ID NO: 27.

  Such modifiers are well known from the prior art (Morita and Jackson, 1986; Skogen et al., 1984, Padmanabhan et al., 1993. J. Mol. Biol., 232: 947-966 or US Patent Application Publication No. 2009 / 229,119). issue).

  The pharmaceutical composition according to the present invention can be formulated as any dosage form necessary for administration. In particular, for systemic administration, the composition according to the invention can be formulated in the form of a lyophilized powder that is sterile for injection. The pharmaceutical composition according to the invention can also be administered nasally or parenterally. Accordingly, the pharmaceutical compositions of the present invention, in addition to the active ingredient, are known to those skilled in the art, and are any pharmaceutically acceptable formulation additives necessary for preparing the desired form of the pharmaceutical composition, in particular Any excipient capable of stabilizing the lyophilized protein GDXa after reconstitution with an aqueous solution for immediate injection can be included.

  In the case of hemorrhagic accidents, the pharmaceutical composition according to the invention can be administered at a concentration 10-20 times lower than that of recombinant FVIIa (Novoseven®), which is 90-270 μg / kg per dose administered. Is administered according to a dose in the range of Thus, according to another aspect, the present invention also provides TFPI to administer 4.5-27 μg / kg per dose to hemophilia A or B patients with or without inhibitors. The present invention relates to a pharmaceutical composition comprising GDXa that can bind to phospholipid but does not have thrombogenicity that cannot bind to phospholipid. The administration can be by systemic, nasal or parenteral routes.

  The invention is illustrated by the following examples.

[Example]
Example 1
Materials and Methods Materials:
Pool of normal patient frozen plasma and individual plasma of hemophilia A or hemophilia B patient, phospholipid TGT, Prionex, corn trypsin inhibitor (CTI), chromogenic substrate PNAPEP 1025, human factor Xa, human GDXa And sheep anti-human thrombin antibody was obtained from Cryopep (Montpellier, France). Human recombinant TFPI is available from Sino Biological Inc. (Beijing, China). Relipidated recombinant human tissue factor (TF, Innovin) is from Siemens Healthcare Diagnostics (Puteaux, France). Thrombin standards, FluCaKit and Diagnostica Stago (Asnieres, France) round bottom 96 well microtiter plates (Immulon 2HB, U bottom plate) were used for thrombin generation studies. For enzyme experiments, flat bottom 96 well microtiter plates are obtained from Greiner (Frickenhausen, Germany) and sheep anti-TFPI antibodies are obtained from Affinity Biologicals (Sandhill Drive, Canada). An Actichrom assay for TFPI activity from American Diagnostica (Stamford, USA) was used to measure TFPI activity. Antithrombin activity assay (STA-Stachrom antithrombin III) is from Diagnostica STAGO. Enzymatic calculations were performed using PRISM 5.0.

Method 1) Thrombin generation assay (TGA)
Measurement of thrombin generation was performed by Hemker's method using tissue factor (TF) 1 pM for blood coagulation activity and CTI 30 μg / ml to inhibit activation of the blood coagulation contact phase during the incubation period. (Van Veen JJ, Gatt A, Cooper PC, Kitchen S, Bowyer AE, Makris M. "In a fluorogenic thrombin generation assay, corn trypsin inhibitor is required only in low concentrations of tissue factor, Factor VIII influences the relationship between factor VIII coagulant activity and thrombogram (Cor trypsin inhibitor in fluorogenic thrombin-generation measurements is only necessary at low tissue factor concentrations and influences the relationship between factor VIII coagulant activity and thrombogram paramete rs.) "Blood Coagul Fibrinolysis. 2008 Apr; 19 (3): 183-9). Briefly, a mixture of 20 μl TF, 4 μM phospholipid and 80 μl plasma was pipetted in triplicate into a microtiter plate. 20 μl of thrombin standard with 80 μl of plasma was also pipetted into the plate in triplicate. The plate was then inserted into a Varioskan (Thermofischer, Illkirch, France) and the excitation wavelength was set to 390 nm, the emission wavelength 460 nm and the passband 10 nm. FluCaKit 20 μl (fluorogenic substrate (Z-Gly-Gly-Arg-AMC, ZGGR-AMC) 2.5 mM and CaCl ₂ 0.1 M) was injected into all wells, thus starting the reaction. The fluorescent signal is read every 20 seconds for 60 minutes. Raw data on fluorescence intensity was exported to Sigmaplot® 9.0 for mathematical calculations using the previously described three wavelength method (De Smedt E. Advanced thromboscopy: PhD thesis, University Maastricht). 2007).

After that
ETP shows the ability to produce endogenous thrombin and corresponds to the area under the curve.

  PH indicates the peak height and corresponds to the peak thrombin level.

  LT represents the latency and corresponds to the time to reach thrombin 2 nM.

  PT is the peak time and corresponds to the time at which PH is obtained.

  Various factors GDXa, Xa or Novoseven® are diluted in buffer A containing pHionex 1%, HEPES 18 mM, sodium chloride 135 mM, pH 7.35 and pretreated with various concentrations of CTI in hemophilic plasma Added.

2) Neutralization of antithrombin and TFPI by specific antibodies To neutralize antithrombin, various concentrations of sheep IgG anti-human antithrombin antibodies (1.8, 3, 5, and 7) in severe hemophilia A plasma. 0.5 g / l) and incubated for 1 hour at 25 ° C. before testing with TGA. In parallel, antithrombin activity was measured with an antithrombin III STA-Statrom reagent on a STAR blood coagulometer (Diagnostica Stago).

  To neutralize TFPI, the same hemophilia A plasma was contacted with various concentrations of sheep anti-human TFPI immunoglobulin (2.5, 5, 10 and 50 mg / l) before testing with TGA. . In parallel, TFPI activity was measured by the Actichrom TFPI activity assay according to the manufacturer's instructions. Briefly, 20 μl of 20-fold diluted plasma was incubated at 37 ° C. in the presence of 20 μl TF / FVIIa for 30 minutes. The factor X (FX) was then added and the whole was incubated at 37 ° C. for 15 minutes before adding EDTA and Spectrozyme FXa. The reaction was stopped after 5 minutes by adding glacial acetic acid and the absorbance was read at 405 nm.

3) Measurement of chromogen 3.1) Measurement of kinetic constants of GDXa and Xa To measure GDXa or FXa activity, 0.3 nM enzyme was added to a buffer containing Prionex 1%, HEPES 18 mM, sodium chloride 135 mM, pH 8 Incubate at 37 ° C for 5 minutes in 4. The chromogenic substrate PNAPEP 1025 is then added at concentrations of 0.33, 0.50, 1, 1.5 and 2.0 mM and the variation in absorption is recorded at 405 nm.

3.2) Enzymatic inhibition of antithrombin (AT) Xa or GDXa (1.25 nM) is incubated in buffer A at 37 ° C. in the presence of increasing concentrations of antithrombin (0-500 nM). . Aliquots of 200 microliters of the mixture are taken at various time intervals up to 90 minutes. The chromogenic substrate PNAPEP 1025 50 μl-6 mM is then added and the change in absorption is recorded.

3.3) Enzymatic inhibition of TFPI Inhibition of GDXa or FXa activity by TFPI was achieved by increasing the concentration of TFPI (0-30 nM for GDXa) in buffer A at 0.25 nM for 3 hours at 25 ° C. And FXa was analyzed by incubating in a final volume of 200 μl in the presence of 0-10 nM). Next, 50 μl of chromogenic substrate PNAPEP 1025 2.5 mM was added and the change in absorption was recorded. Ki ^* was measured as described above (Bunce MW, Toso R, Camire RM. Enzyme-like factor Xa variants restore thrombin production and effectively bypass the endogenous pathway in vitro (Zymogen- like factor Xa variants restore thrombin generation and effectively bypass the intrinsic pathway in vitro) Blood.2011 Jan 6; 117 (1): 290-8; Bough RJ, Broze GJ, Jr., Krishnaswamy S. Exogenous by tissue factor pathway inhibitor Regulation of extrinsic pathway factor Xa formation by tissue factor pathway inhibitor. J Biol Chem. 1998; 273 (8): 4378-86).

3.4) Measurement of plasma half-life of GDXa and Xa The plasma half-life of GDXa or Xa was measured by adding GDXa or Xa 50 nM to normal plasma. The mixture was then incubated at 37 ° C. A small amount was collected from 0 to 60 minutes and immediately diluted 25-fold with buffer A before adding the chromogenic substrate PNAPEP 1025 1.5 mM as described previously and measuring the remaining amidolytic activity.

Example 2
Preparation of Modified Factor Xa Plasmid pTT5 was cleaved by digestion with HindIII-BamHI enzyme and encoded a signal peptide of TIMP1 with HindIII and NheI restriction sites and FX lacking the Gla domain with NheI and BamHI restriction sites. The gene to be inserted is inserted to generate plasmid pTT5-TIMX (pTT5 spTIMP1 gla deficient FX). A sequence encoding FX according to the present invention with NheI and BamHI restriction sites and lacking the Gla domain was obtained by chemical synthesis (GenScript Corporation). Next, a furin recognition site was introduced upstream of the heavy chain N-terminal isoleucine, allowing GDXa to be secreted directly into the medium for purification. The generated GDXa is represented by the sequence of SEQ ID NO: 3 for the light chain and SEQ ID NO: 2 for the heavy chain.

Example 3
Measurement of GDXa and FXa Kinetic Parameters Before analyzing the effect of GDXa on thrombin generation, cleavage of the chromogenic substrate PNAPEP 1025 was used to characterize GDXa and FXa. GDXa has similar affinity (Km = 0.75 ± 0.05 mM) to FXa (Km = 0.64 ± 0.03 mM), and similar catalytic properties: GDXa has kcat = 290 ± 5 s-1 and FXa has kcat = 375 ± 8 s −1 was shown (Table 1). These results were previously reported with the chromogenic substrate S2222 (Skogen WF, Esmon CT, Cox AC. “Comparison of factor Xa coagulation factor and des- (1-44) factor Xa in the association of prothrombinase ( Comparison of coagulation factor Xa and des- (1-44) factor Xa in the assembly of prothrombinase) "J Biol Chem. 1984; 259 (4): 2306-10).

  The results shown correspond to two independent measurements performed in triplicate using the chromogenic substrate PNAPEP 1025.

Example 4
Effect of GDXa or FXa on thrombin generation At a concentration of 1 pM, TF is unable to induce thrombin generation in severe hemophilic plasma A, as shown by the dotted line in FIG. However, in the presence of GDXa50pM, a clear recovery of thrombin generation was observed (FIG. 1B). GDXa restores all parameters involved in thrombin generation, such as endogenous thrombin production capacity (ETP), latency, peak height, peak time (FIG. 1B, Table 4). The observed thrombin generation was not a direct effect of GDXa on plasma since thrombin was not generated without TF (FIG. 1B, dotted line). This was observed in all hemophilia plasmas tested. Furthermore, in contrast to GDXa, FXa induced thrombin generation in the absence of TF because it can directly convert prothrombin to thrombin (FIG. 1C). To quantify the possibility of interference by direct cleavage of the substrate ZGGR-AMC by GDXa, increasing amounts of enzyme were added in the presence of a saturating amount of repirudin (thrombin inhibitor) (6 pg / ml). As shown in FIG. 1D, the raw fluorescent signal was completely extinguished in the presence of lepirudin. Under these conditions, GDXa cleaved the substrate ZGGR-AMC in proportion to concentration. At a concentration of 50 nM, the final signal corresponded to 8% of the fluorescence generated without lepirudin, and at a concentration of 250 nM, the signal showed about 40% of the total fluorescence. However, since this signal is linear, it is mathematically included in the complex α2-macroglobulin-thrombin signal in the three-wavelength method used to calculate the thrombin concentration curve and does not affect the results of thrombin generation. (De Smedt E. Advanced thrombinoscopy: PhD thesis, University Maastricht; 2007).

  The minimum amount of GDXa that could restore thrombin generation in the plasma of patients with severe hemophilia A was evaluated. A concentration of 20 nM GDXa gave a thrombin generation curve in this plasma similar to that observed in normal plasma (FIG. 1A) (FIG. 1E). Furthermore, 10 nM GDXa produced a slightly higher signal than that obtained in the presence of 200 nM rFVIIa (FIG. 1F).

Example 5
Effects of anti-antithrombin and anti-TFPI antibodies on thrombin generation Anti-antithrombin antibodies can greatly increase ETP with little effect on kinetic parameters (Table 2 and Figure 2A). With an anti-antithrombin antibody 7.5 g / l (residual antithrombin activity 9%), ETP was 2716 nM. The minute rose and the PH reached 76 nM. This is in contrast to the effect on kinetic parameters (LT = 6.6 minutes, PT = 31.3 minutes). In addition, as already shown by Erhardtsen et al. ("Blocking of tissue factor pathway inhibitor (TFPI) shortening) The bleeding time in rabbits with antibody induced hemophilia A.) "Blood Coagul Fibrinolysis. 1995; 6 (5): 388-94), anti-TFPI antibodies were also able to restore blood coagulation in hemophilic plasma . Anti-TFPI antibodies at concentrations above 10 mg / l (residual TFPI activity <30%) normalized all TGA parameters in this hemophilic plasma (Table 2). At 10 mg / l (Table 2 and FIG. 2B), ETP increased from 209 to 762 nM and PH increased from 8 to 79 nM, respectively. LT decreased from 13.6 to 3.5 minutes and PT decreased from 25.9 to 7.2 minutes.

  Various concentrations of human antithrombin antibody or anti-human TFPI antibody were added to the plasma of patients with severe hemophilia A prior to the thrombin generation assay.

HP: hemophilia plasma;
NP: normal plasma.

  Antithrombin and TFPI residual activity were measured according to the methods described in Materials and Methods.

  GDXa: hemophilic plasma with GDXa 50 nM.

  The concentration of anti-ATIgG is expressed in g / l.

  The concentration of anti-TFPI antibody is expressed in mg / l.

Example 6
Enzymatic inhibition of GDXa and FXa by TFPI and antithrombin For irreversible enzymatic inhibition by antithrombin, GDXa (1.50 ± 0.04 × 103 M ⁻¹ .s ⁻¹ , FIG. 2B) and inhibition of Xa The characteristics were the same (k2 = 1.57 ± 0.08 × 103 M ⁻¹ .s ⁻¹ , FIG. 2A). TFPI is a slow fixation inhibitor of FXa (26, 27), and at low concentrations it is a weak inhibitor of GDXa (28-30). Therefore, the inhibition of FXa and GDXa was compared (Table 1). GDXa showed a lower affinity for TFPI (Ki ^* = 0.31 ± 0.04 nM, FIG. 2D) compared to FXa (Ki ^* = 0.17 ± 0.03 nM, FIG. 2C). Furthermore, the achievement of equilibrium in this experiment was shown by checking the titration curve after 18 hours of incubation.

Example 7
Plasma half-life of GDXa and Xa Considering inhibition of GDXa by TFPI and antithrombin, residual activity after addition of GDXa or FXa 50 nM to plasma at 37 ° C. was evaluated. As shown in FIG. 4A, plasma activity decreased rapidly, with a half-life of approximately 1 minute 30 seconds, as previously shown with FXa (Bunce MW, Toso R, Camire RM. -Like factor Xa variants restore thrombin generation and effectively bypass the in vitro intrinsic pathway "Blood. 2011 Jan 6; 117 (1): 290-8), GDXa or FXa reached a plateau after 20 minutes. Nevertheless, the effect on thrombin generation is maintained over time, for example, after 1 hour, the remaining activity of GDXa is about 10% of the original activity (FIG. 4B) and the recovery of thrombin generation is maintained. It had been. After 1 minute incubation, ETP increased to 0-610 nM and was still 478 nm after 60 minutes (Table 3). Similar normalization was also observed for maximum peak height (75 nM at 1 minute, 38 nM at 60 minutes) and shift and peak times (Table 3).

  GDXa 50 nM was added to severe hemophilic plasma and incubated at 37 ° C. for 1 hour. Small amounts were taken immediately and after 1 hour to measure thrombin production.

Example 8
Effect of GDXa on thrombin generation in patients with severe hemophilia A with and without inhibitors and patients with severe hemophilia B Thrombin generation is a severe condition involving one donor whose inhibitor titer was measured at 50 Bethesda units. Plasma samples from 5 different donors with hemophilia A and 1 plasma from patients with severe hemophilia B were evaluated. Thrombin generation was hardly detectable in 6 plasmas when blood clotting was induced by TF1pM but recovered in the presence of GDXa20 and 50 nM. Table 4 shows that normalization is observed to varying degrees for all plasma and all parameters related to thrombin generation. In the presence of GDXa 20 nM, the observed ETP was 374 ± 128 nM and the PH was 22 ± 11 nM. LT decreased to 5.0 ± 1.5 and PT decreased to 13.9 ± 3.8 minutes.

Furthermore, a dose effect was observed, and when GDXa 50 nM was added, the value increased to 533 ± 132 nM for ETP and 46 ± 20 nM for PH. LT decreased to 2.8 ± 0.7 and PT decreased to 9.2 ± 2.7 minutes.

  GDXa20 or 20 nM was added to various plasmas and immediately assayed for thrombin generation.

Px: Plasma x
HA: hemophilia A
HA + I: hemophilia A with inhibitor (hemophilia A with inhibitor 50BU)
HB: Hemophilia B
Example 9
Comparison of GDFXa according to the present invention with Factor VIIa (rVIIa, Novoseven®) for thrombin generation in hemophilia A subjects FIG. 5 shows that GDXa is much more thrombin-generated than rVIIa (Novoseven®). It is effective for normalization.

“ALJAMALI MN, KJALKE M, HEDNER U, EZBAN M, TRANHOL M.” Thrombin generation induced by rFVIIa (NovoSeven®) and NN1731 in a reconstituted cell-based model that mimics the hemophilia state platelet activation (Thrombin generation and platelet activation induced by rFVIIa (NovoSeven (R)) and NN1731 in a reconstituted cell-based model mimicking hemophilia conditions.) "Haemophilia. 2009; 15: 1318-26 ", at least 500 nM of rVIIa is required for normalization.

The figure which shows the influence of GDXa or FXa with respect to the production | generation of thrombin.

  (A) Thrombin generation test in the presence of phospholipids and TF in a pool of normal plasma (solid line) and a pool of severe hemophilia A plasma (dotted line). (B) GDXa-rich (50 nM) hemophilic plasma with TF in the presence of phospholipids (solid line) and without TF (dotted line). (C) hemophilia plasma rich in FXa (50 nM) with TF in the presence of phospholipids (solid line) and without TF (dotted line). (D) Cleavage of substrate ZGGR-AMC by GDXa. Tested with and without repirudine 6 pg / ml (control curve A), in the absence of GDXa (curve B) or in the presence of various concentrations (curves CE) in the presence of phospholipids and TF. CTI pretreated normal plasma. (E) Thrombin in the presence of phospholipids and TF in hemophilic plasma enriched with GDXa 10 nM (solid line) or 20 nM (dotted line) and (F) rFVIIa 40 nM (bold line) or 200 nm (dotted line) Generation.

The data represent experiments performed on at least three different plasmas of various severe type A hemophilia.
The figure which shows the influence of the anti- AT and anti- TFPI antibody with respect to the thrombin production | generation in the plasma of hemophilia.

  Various concentrations of anti-human antithrombin (A) or anti-human TFPI (B) antibody were added to severe hemophilia plasma A and then left for thrombin generation. The antibody concentration is expressed in g / l for anti-antithrombin antibody and in mg / l for anti-TFPI antibody. The concentration of GDXa is nM. HP indicates hemophilic plasma and NP indicates normal plasma.

(A) Curve A shows hemophilia plasma, curves BD show antithrombin antibodies at various concentrations added to hemophilia plasma, and curve E added to hemophilia plasma GDXa 50 nM added, curve F shows normal plasma. (B) Curve A shows hemophilia plasma, curves BD show anti-TFPI antibodies at various concentrations added to hemophilia plasma, and curve E added to hemophilia plasma GDXa 50 nM added, curve F shows normal plasma.
The figure which shows the enzymatic inhibition of GDXa and FXa by TFPI or antithrombin.

  (AB) For the inhibition of antithrombin, presence of 1.25 nM FXa (A) or (B) GDXa at increasing concentrations of antithrombin (AT: 0 (curve A) to 500 nM (curve F)) Incubated at 37 ° C under. Small amounts were collected at various time points (0-90 minutes) and added to the chromogenic substrate PNAPEP 1025. (C-D) Inhibition of FXa (C) or GDXa (D) activity by TFPI in enzyme A in the presence of increasing concentrations of TFPI (GDXa 0-30 nM and FXa 0-10 nM) in buffer A. Analyzed by incubation with 25 nM at 25 ° C. for 3 hours.

The data represents two different experiments.
The figure which shows the measurement of the pharmacokinetic characteristic of GDXa and FXa in plasma.

  50 nM FXa (A) or GDXa (B) was added to normal plasma and the whole was incubated at 37 ° C. Small amounts were taken at different time intervals and immediately diluted 25-fold with buffer A to measure residual activity. The half-life was 1.4 ± 0.1 minutes for FXa and 1.8 ± 0.1 minutes for GDXa.

Data is the average of two different experiments.
The figure which shows normalization of the thrombin production by rVIIa and GDXa.

  Add GDXa (curves C and D) or rVIIa at various concentrations (curves E and F) to hemophilic plasma (HP, curve B). NP: normal plasma (curve A).

Claims (8)

  A pharmaceutical composition comprising modified factor Xa (GDXa) for preventing or treating hemorrhagic accidents in patients with hemophilia A or B with or without inhibitors, said GDXa lacking the Gla domain A pharmaceutical composition that is FXa.   2. The composition of claim 1, wherein the GDXa comprises an EGF1 domain defect, an EGF2 domain defect, or an EGF1 and EGF2 domain defect.   The composition according to claim 1 or 2, wherein the GDXa has a complete heavy chain.   Said GDXa is represented by SEQ ID NO: 7 or SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 for the light chain and SEQ ID NO: 2, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 for the heavy chain. The composition according to any one of claims 1 to 3, which is represented by SEQ ID NO: 12, SEQ ID NO: 13 or SEQ ID NO: 26.   Said GDXa is encoded by the nucleic acid sequence of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19 for the light chain, and SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 22, for the heavy chain. The composition according to any one of claims 1 to 3, which is encoded by the nucleic acid sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 27.   2. GDXa for administration by systemic, nasal, or parenteral route to patients with hemophilia A or B with or without inhibitors, comprising 4.5 to 27 μg / kg. The pharmaceutical composition as described in any one of -5.   A method of prophylactically treating hemorrhagic syndrome in patients with hemophilia A or B by administering modified factor Xa (GDXa) or a pharmaceutical composition according to any one of claims 1-5.   A method of treating a hemorrhagic accident in a patient with hemophilia A or B by administering a modified factor Xa (GDXa) or a pharmaceutical composition according to any one of claims 1-5.